It has been a longstanding objective in the art to be able to separate a specific group of microorganisms from a sample. Traditionally, a sample that contains a plurality of microorganisms is diluted and applied to a nutrient rich culture media. Due to the high dilution of the original sample, individual microorganisms are effectively isolated from other microorganisms that were also present in the sample. Over time, colonies of bacteria grow on the media—each colony originating from one of the individual microorganisms. Since each colony shares the same progenitor, such colonies contain only a single type of microorganism. Such colonies may be collected and used to generate substantial quantities of essentially pure microorganisms.
For such a technique to function, the microorganism must be culturable. For reasons that are not clear, the vast majority of microorganisms fail to grow when they are removed from their natural environment. It appears that there is a synergistic relationship between many neighboring species of microorganisms that prevents many species of microorganism from being cultured in isolation. This is a matter of great concern for microbiologists. Often, a scientist wishes to generate antibodies that are specific to a given species of microorganism. However, when the scientist is unable to isolate the microorganism from its environment, the scientist is unable to generate such antibodies.
Cell enrichment/purification approaches have been reported in the literature, but all have significant limitations which hinder their successful application to uncultured microorganisms. Immunomagnetic separation has been shown to be an efficient and sensitive method for the enrichment and isolation of cells in a wide variety of medical and food samples and few environmental samples. See Biotechnology Progress, Vol. 18, (April, 2002), pp. 212-220. In this technique, paramagnetic beads coated with antibodies specific to surface antigens of the target cells are used to label these cells and subsequently separate them from unlabelled cells in a magnetic field. While effective in the medical field, a significant drawback of this method is the requirement for antibodies that are specific to the target organisms. Production of such specific antibodies requires a pure sample of the organism itself. Such pure samples are often not available using conventional techniques.
Optical trapping and manipulation of bacterial cells and viruses with infrared lasers can be applied to collect individual cells. However, this method is time consuming and gives only a small amount of cellular material for analysis. See Review of Scientific Instruments, Vol. 75, (September, 2004), pp. 2787-2809.
Flow cytometry is another method that has been applied to enrich cells. To date, the inventor is aware of only one report showing the enrichment of hybridized bacteria from sediment using flow cytometry. Such a technique is illustrated in a paper to Kalyuzhnaya et al. See Applied & Environmental Microbiology, Vol. 72, (June, 2006), pp. 4293-4301. Kalyuzhnaya et al. enriched hybridized Type I and Type II methanotrophs from Lake Washington sediment. Abundances of target cells in lake sediment samples were 4.7% (percent of total cell counts, Type I methanotrophs) and 1.2% (Type II methanotrophs). After flow cytometric sorting, Type I methanotrophs comprised 59% of the clone library (no cell counts were performed) and Type II methanotrophs counted 47.5%. Another report (Applied & Environmental Microbiology, Vol. 70, (October, 2004), pp. 6210-6219) shows sorting of bacterial groups from plankton after CARD-FISH with purities ranging from 96% to 97%. Flow sorting of activated-sludge microorganisms, hybridized with fluorescein labeled oligonucleotide probes, has been reported by Wallner et al., (Applied and Environmental Microbiology, Vol. 63, (November, 1997), pp. 4223-4231. Here, Acinetobacter was enriched from 0.3 to 0.6% total abundance in the original sample to 35 to 84% in the sorted fraction. Leptotrix was enriched from 12 to 13% in the original sample to 69 to 82% in the sorted fraction.
Fluorescence in situ hybridization (FISH) is commonly used in microbial ecology studies to visualize microorganisms, most often directed toward the 16S rRNA. Until recently, FISH has been limited to the detection of highly expressed rRNA genes. To overcome this limitation, Pernthaler et al. (Applied & Environmental Microbiology, Vol. 68, (June, 2002), pp. 3094-3101) described an adaptation of FISH called CARD-FISH that uses fluorescently labeled tyramides as a substrate for the probe-delivered horseradish peroxidase. Using CARD-FISH the hybridization signal can be increased up to 1000-fold and even slow growing microbes from deep subsurface samples (Nature, Vol 433, (February, 2005), pp. 861-864) can be detected. This method has also recently been applied in a modified protocol for the FISH detection of messenger RNA. See Pernthaler, A., and Pernthaler, J. “Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes.” in: Leadbetter, J. R. (ed)., Methods in Enzymology (San Diego: Elsevier, 2005), pp. 352-371.
Complementary polyribonucleotide probes have been used in conjunction with paramagnetic substrates by Stoffels et al. to separate one species from an artificially produced mix of species (Environmental Microbiology, Vol. 1, (March, 1999), pp. 259-271). Stoffels et al., used polyribonucleotide probes of about 300 bases length, targeting a highly variable region in domain III of the 23S rRNA. The probes of Stoffels have significant limits when compared to oligonucleotide probes: (A) the database for 23S rRNA sequences is much smaller (about 20,000 sequences) than the 16S rRNA database (about 500,000 sequences), thus the possibility to design probes that cross-react with other species is relatively high; (B) the length of these probes complicates probe design; and (C) these probes are RNA probes, which makes them highly susceptible to degradation by RNases, which are present in every sample and every microbe and even survive fixation.
Therefore, a process for separating a desired group of microorganisms from a plurality of microorganisms is desired.